The increasing emergence of antibiotic resistant bacteria is a global problem. [Fauci, A. S., Touchette, N. A. & Folkers, G. K. (2005). Emerging infectious diseases: a 10-year perspective from the national institute of allergy and infectious diseases. Emerg Infect Dis 11, 519-25.] Antibiotic resistant bacteria were responsible for 17 million deaths world-wide in 1996 with an estimated cost of $30 billion dollars in the United States alone. [Levy, S. B. & Marshall, B. (2004). Antibacterial resistance worldwide: causes, challenges and responses. Nat Med 10, S122-9.] Funding for new research by major pharmaceutical companies has steadily decreased, as has the development of new antimicrobials. Furthermore, the rapid emergence of resistance to new classes of antimicrobials has limited their use in clinical settings. These trends, if continued, will result in a lack of effective antimicrobials for a majority of bacterial infections in the years to come.
The mechanism of resistance for all currently used therapeutics has been determined. There are several general mechanisms of bacterial resistance: 1) reduction of antibiotic uptake, 2) transport of the antibiotic out of the cell, 3) enzymatic inactivation of the antibiotic, 4) use of an alternative metabolic pathway, 5) titration of the antibiotic by overproduction of the target, and 6) target modification so that it is no longer recognized by the antibiotic. [Laios, E., Waddington, M., Saraiya, A. A., Baker, K. A., O'Connor, E., Pamarathy, D. & Cunningham, P. R. (2004). Combinatorial genetic technology for the development of new anti-infectives. Arch Pathol Lab Med 128, 1351-9.] Of these mechanisms, target modification is the most common mechanism of resistance for newly developed antibiotics. The specificity of antibiotic—target binding involves the structure as well as the sequence of the target. Mutations that affect the sequence or structure of the target without effecting function may reduce or eliminate antibiotic binding and result in resistance. For example, aminoglycoside antibiotics target the A-site of bacterial 16 S ribosomal RNA and increase the translational error rate. [Magnet, S. & Blanchard, J. S. (2005). Molecular insights into aminoglycoside action and resistance. Chem Rev 105, 477-98.] A single A1408G mutation reduces ribosome function by approximately 30% (unpublished results) but completely disrupts binding of certain aminoglycoside antibiotics. [Recht, M. I., Douthwaite, S., Dahlquist, K. D. & Puglisi, J. D. (1999). Effect of mutations in the A site of 16 S rRNA on aminoglycoside antibiotic-ribosome interaction. J Mol Biol 286, 33-43.] Therefore, targeting an antibiotic to all possible mutants of a particular ribosomal region that maintain function would eliminate this mechanism of resistance.
Nearly half of all naturally occurring antibiotics target an aspect of protein synthesis and more specifically the ribosome. The 70 S bacterial ribosome is responsible for the translation of messenger RNA (mRNA) into protein. Ribosome crystal structures and biochemical studies have shown that the RNA is the catalytically active component of the ribosome, therefore, the ribosome is a ribozyme. [Yusupov, M. M., Yusupova, G. Z., Baucom, A., Lieberman, K., Earnest, T. N., Cate, J. H. & Noller, H. F. (2001). Crystal structure of the ribosome at 5.5 A resolution. Science 292, 883-96; Wimberly, B. T., Brodersen, D. E., Clemons, W. M., Jr., Morgan-Warren, R. J., Carter, A. P., Vonrhein, C., Hartsch, T. & Ramakrishnan, V. (2000). Structure of the 30S ribosomal subunit. Nature 407, 327-39.] The essential nature of the protein synthesis process makes the ribosomal RNA (rRNA) an ideal drug target.
Studies of the rRNA sequences from numerous different organisms have shown that the overall structure of the ribosome is conserved within all three domains of life. Phylogenetic analysis of rRNA sequences has provided much information about pairing interactions and nucleotide conservation. Each of these analyses, however, employs genomic or organelle rRNA sequences. These sequences are constrained by their essential role in protein synthesis. As a result, very little or no sequence variation is observed in rRNA regions believed to be functionally important, since even subtle changes to the structure surrounding critical residues may reduce function and affect fidelity. Additionally, these conserved sites may be structurally important rather than functionally important. Therefore, drugs that target these sites would allow for resistance if the sequence can mutate but maintain the functional structure. An ideal drug would target all possible functional mutations at the target site.